1. Field of the Invention
This invention relates to a method for attaching and detecting the attachment of biological molecules to semiconductors, and more particularly, the invention relates to a method for attaching biologically active molecules to nanoparticle-size metal oxide semi-conductors.
2. Background of the Invention
Detection of target molecules in an unknown mixture finds a variety of applications. A few of these applications include genome sequencing, forensics, assays, and drug affinity studies.
Typical detection techniques involve the use of fluorescence tags. Such tags are first attached to moieties (having known affinities to target molecules), to create a construct. The construct is then combined with materials in a search for the target molecules suspected of residing in those materials.
A myriad of problems exist with the use of fluorescence molecules in detection schemes. For example, the tags tend to fluoresce at a wide wavelength band and therefore obliterate the xe2x80x9cfingerprintxe2x80x9d of other concomitantly used fluorescent tags.
Also, fluorescence moieties are short-lived, particularly at wavelengths required to induce fluorescence. As such, exposure times to the wavelengths must often be minimized. Strict ambient conditions also are required to forestall eventual tag denigration.
Efforts have been made to eliminate fluorescence tag usage in detection processes. For example, U.S. Pat. No. 5,990,479 awarded to Weiss et al on Nov. 23, 1999, supplants fluorescence moieties with semiconductor moieties. The semiconductors are attached to affinity molecules to create a construct which in turn is mixed with material suspected of containing target molecules. Detection is noted when the mixture is subjected to light at wavelengths which cause the semi-conductor in the construct to luminesce.
The ""479 patent eliminates many of the drawbacks of some fluorescence systems. For example, each semiconductor imparts luminescence at narrow band wavelengths. This feature allows several semiconductors, each with characteristic emission spectra, to be used simultaneously to detect several different target molecules.
However, state of the art semiconductor detection systems do not provide a means for determining the amount of target moiety detected. Also, detection sensitivities are limited to optical characteristics of the semiconductor.
Notwithstanding the foregoing drawbacks in semiconductor systems, the inventors envision exploiting the phenomenon in those substrates whereby radical intermediates are formed following light induced charge pair formation. Electron Paramagnetic Resonance (EPR) is the prime technique for detecting these formations.
Nanocrystalline metal oxide semiconductor particles that are durable and are not susceptible to photo-degradation, act as miniaturized electrochemical cells and act as stable and efficient artificial photosynthetic systems. However, the recombination kinetics in these systems is very fast, on the order of picoseconds. N. Serpone et al. J. Phys. Chem., 99, 16655 (1995). Unless the charge separation is increased by reaction with adsorbed species, the efficiency of charge separation is very low.
A need exists in the art for a detection system based on electronic changes in a foundation substrate. The system should incorporate a means to modify the charge separation tendencies of photo-induced ion pairs on the substrates so as to be measurable with existing time-resolved detection systems. This modification would allow chemical reactions to be efficiently performed using nanocrystalline materials. The system should also serve as a detector for the existence of moieties that would modify the charge separation fingerprint via electron donation or extraction. The charge separation should be detectable via electronic signals and easily amplified.
An object of the present invention is to provide a method for detecting molecules that overcomes many of the disadvantages of the prior art.
Another object of the present invention is to provide a method for exploiting the charge separation abilities of semi-conductors to create detectors of molecules. A feature of the invention is the utilization of a bidentate or tridentate modifier molecule as an electron donor or acceptor to the semiconductor. An advantage of the invention is the prolongation of charge separation on the semi-conductor as an indicator of the type of molecule juxtaposed and electrically connected to the semi-conductor particle.
Yet another object of the present invention is to provide a method for selective binding and modifying of molecules in vivo or in vitro. A feature of the method is that a nanocrystalline-biological construct, capable of photochemical response, and capable of simultaneously carrying a number of biologically active molecules, can bypass biological membranes via standard delivery methods. An advantage of the method is that oxidative damage, produced by positive charge centers (resulting in the formation of oxygen centered radicals covalently linked to surface semi-conductor atoms), facilitates the cleaving and recombination of particle-attached molecules to reactive sites. Another advantage is that a collection of biologically active molecules can be delivered to, and therefore co-localized at, the reactive sites to facilitate simultaneous action of the delivered biomolecules.
Still another object of the present invention is to provide a molecule detection system which also quantifies the amount of target molecule present. A feature of the present invention is the utilization of a nanocrystalline foundation material capable of binding a plurality of linker moieties, whereby the moieties link the material to the target molecule. An advantage of the invention is that the detector construct facilitates selective adsorption and selective chemical reactions at the surface of the material.
Briefly, the invention provides for a method for detecting molecules, the method comprising determining the electronic status of a semi-conductor; establishing electronic communication between the molecules and the semiconductor; subjecting the semi-conductor to energy influx; and redetermining the electronic status of the semi-conductor.
Also provided is a method for detecting biological molecules, the method comprising supplying a semi-conductor having a first energy level and a second energy level and whereby the first energy level corresponds to a first optical characteristic of the semi-conductor; establishing electrical contact between the semi-conductor and the molecules; causing electrons to move from the molecules to the second energy level; and monitoring any change in the first optical characteristic.
Also provided is a method for detecting target moieties in situ, the method comprising binding biological material to nanocrystalline semiconductor particles, wherein the material has an affinity to the target moiety; facilitating entry of the bound material into an organelle; and subjecting the semiconductor to radiation sufficient to produce a charge pair separation on the semiconductor""s surface.